Báo cáo khoa học: Silencing the constitutive active transcription factor CREB by the LKB1-SIK signaling cascade pot

Silencing the constitutive active transcription factor CREB
by the LKB1-SIK signaling cascade
Yoshiko Katoh
1
, Hiroshi Takemori
2
, Xing-zi Lin
1
, Mitsuhiro Tamura
1
, Masaaki Muraoka
3
,
Tomohiro Satoh
3
, Yuko Tsuchiya
4
,LiMin
1
, Junko Doi
5
, Akira Miyauchi
3
, Lee A. Witters
6
,
Haruki Nakamura
4
and Mitsuhiro Okamoto
7
1 Molecular Physiological Chemistry, Osaka University Medical School, Japan

2 Laboratory of Cell Signaling and Metabolism, National Institute of Biomedical Innovation, Osaka, Japan
3 ProteinExpress Co. Ltd, Chiba, Japan
4 Institute for Protein Research, Osaka University, Japan
5 Food and Nutrition, Senri Kinran University, Osaka, Japan
6 Departments of Medicine and Biochemistry, Dartmouth College, Hanover, NH, USA
7 Department of Food and Nutrition, Tezukayama University, Nara, Japan
Cyclic AMP-responsive element (CRE)-binding protein
(CREB) is a transcription factor that plays an import-
ant role in numerous physiological events, such as cell
proliferation, survival, tumorigenesis, glucose metabo-
lism and memory, in a phosphorylation-dependent
manner [1,2]. Upstream signals arriving at CREB are
Keywords
cAMP responsive element; CRE-binding
protein; LKB1; salt-inducible kinase;
transducer of regulated CREB activity
Correspondence
H. Takemori, Laboratory of Cell Signaling
and Metabolism, National Institute of
Biomedical Innovation, 7-6-8, Asagi, Saito,
Ibaraki, Osaka, 567-0085, Japan
Fax: +81 72 641 9836
Tel: +81 72 641 9834
E-mail:
(Received 16 January 2006, revised 19 April
2006, accepted 24 April 2006)
doi:10.1111/j.1742-4658.2006.05291.x
Cyclic AMP responsive element (CRE)-binding protein (CREB) is known
to activate transcription when its Ser133 is phosphorylated. Two independ-
ent investigations have suggested the presence of Ser133-independent acti-

vation. One study identified a kinase, salt-inducible kinase (SIK), which
repressed CREB; the other isolated a novel CREB-specific coactivator,
transducer of regulated CREB activity (TORC), which upregulated CREB
activity. These two opposing signals are connected by the fact that SIK
phosphorylates TORC and induces its nuclear export. Because LKB1 has
been reported to be an upstream kinase of SIK, we used LKB1-defective
HeLa cells to further elucidate TORC-dependent CREB activation. In the
absence of LKB1, SIK was unable to phosphorylate TORC, which led to
constitutive activation of CRE activity. Overexpression of LKB1 in HeLa
cells improved the CRE-dependent transcription in a regulated manner.
The inactivation of kinase cascades by 10 nm staurosporine in LKB1-posit-
ive HEK293 cells also induced unregulated, constitutively activated, CRE
activity. Treatment with staurosporine completely inhibited SIK kinase
activity without any significant effect on the phosphorylation level at the
LKB1-phosphorylatable site in SIK or the activity of AMPK, another tar-
get of LKB1. Constitutive activation of CREB in LKB1-defective cells or
in staurosporine-treated cells was not accompanied by CREB phosphoryla-
tion at Ser133. The results suggest that LKB1 and its downstream SIK
play an important role in silencing CREB activity via the phosphorylation
of TORC, and such silencing may be indispensable for the regulated activa-
tion of CREB.
Abbreviations
A-loop, activation loop; AMPK, AMP-activated protein kinase; bZIP, basic leucine zipper domain; CRE, cAMP-response element; CREB,
CRE-binding protein; DAPI, 4¢,6-diamidino-2-phenylindole; GFP, green fluorescent protein; GST, glutathione-S-transferase; HA, hemagglutinin;
KID, kinase-inducible domain; moi, multiplicities of infection; PKA, protein kinase A; RT, reverse transcription; SIK, salt-inducible kinase;
TORC, transducer of regulated CREB activity.
2730 FEBS Journal 273 (2006) 2730–2748 ª 2006 The Authors Journal compilation ª 2006 FEBS
conveyed to transcriptional machineries via two distinct
domains of CREB. The kinase-inducible domain (KID)
is located in the N-terminal region, which contains an

activating phosphoacceptor residue, Ser133. The other
domain in the C-terminus is composed of a basic leu-
cine zipper (bZIP) that is responsible for dimerization
and binding to the CRE. Phosphorylation of Ser133
alters the affinity of KID to the KIX domain of CREB
and p300, resulting in enhanced transcription of CRE-
dependent genes [3]. Development of a specific anti-
body that recognized phospho-Ser133 [4,5] enabled
investigators to monitor the level of ‘activated CREB’,
which has now significantly accelerated the studies of
phosphorylation-dependent CREB activation.
Possible involvement of the bZIP domain of CREB
in the regulation of CRE-dependent gene expression
has been suggested by the results of two lines of
research. One was initiated by an mRNA subtraction
study to isolate a specific molecule induced in the adre-
nal gland under the stress of consuming a high-salt diet.
The molecule isolated was a kinase, and, thus, we
named it salt-inducible kinase (SIK) [6]. SIK is a mem-
ber of the AMP-activated protein kinase (AMPK) fam-
ily [7]. A gene database search found three isoforms of
SIK, SIK1–3 [8,9]. In Y1 mouse adrenocortical tumor
cells, levels of mRNA, protein and kinase activity of
SIK1 were elevated within 30 min after the initiation of
the cAMP–protein kinase A (PKA) cascade. Overex-
pression of SIK1 inhibited gene expression(s) induced
by cAMP [10]. Analyses of the promoter regions of
such genes indicated that CREs in the promoters were
the sites where SIK-mediated transcriptional repression
occurred, and, thus, SIK1 was thought to repress

CREB activity [11]. Although SIK seemed not to phos-
phorylate CREB directly, it repressed CREB in a
kinase-activity-dependent manner. Mapping the region
where SIK1 exerted its repressive action suggested that
SIK1 repressed CREB by acting on the bZIP domain
[11]. In reporter gene assays, overexpression of the kin-
ase domain of SIK1 repressed CRE-dependent tran-
scription completely, even when CREB was supposed
to be fully activated by overexpression of PKA. We
therefore thought that the phosphorylation of CREB at
Ser133 was not sufficient for making ‘activated CREB’.
The second line of research began in an attempt to
isolate novel factors that could modulate CREB activ-
ity by using high-throughput transformation assays
[12,13]. Expression vectors containing full-length
cDNAs were cotransformed with reporter vectors in
HEK293 cells, and a new family of coactivators was
identified. They were named transducer of regulated
CREB activity (TORC) 1–3. The N-terminal region
of TORCs formed a coiled-coil structure, which
interacted with the bZIP domain of CREB [12]. Once
the TORCs had been overexpressed in HEK293 cells,
CREB-dependent transcriptions were upregulated at,
or beyond, the levels induced by cAMP. Activation of
CREs by an overexpression of TORCs required
CREB-, but not Ser133 phosphorylation, and, thus,
TORCs were thought to be coactivators that did not
require CREB phosphorylation. Cytochemical studies
of TORCs, however, showed that the activating signals
that could phosphorylate CREB, such as cAMP and

Ca
2+
, also induced the nuclear import of TORCs
[14,15], suggesting that combination of Ser133 phos-
phorylation and the binding of TORCs to the bZIP
domain produces the fully ‘activated CREB’.
The above observations indicate that SIKs and
TORCs share a common feature regarding the regula-
tion of CREB activity, both acting on the bZIP domain
of CREB in a phospho-Ser133-independent manner.
Having examined this feature further, we found that
SIK2 phosphorylated TORC2 at Ser171. The resulting
phospho-TORC2 was exported from the nucleus to the
cytoplasm, and this led to the apparent inactivation of
the CREB activity [14]. We also showed that PKA
phosphorylated SIK1 [16] and the phospho-SIK1 could
not induce the nuclear export of TORC2 [17].
The importance of the phospho ⁄ dephospho regula-
tion of TORC, by regulating CREB, was also shown as
a physiological impact in hepatic gluconeogenesis [18].
However, it remains to be clarified as to whether the
phospho ⁄ dephospho regulation of TORC is one of
the regulatory mechanisms for the CREB activity or
the cascade indispensable for the CREB action. AMPK
family kinases, including SIK, have flexible activation
loops (A-loops) near their substrate-binding pockets.
Phosphorylation in the A-loop induces a structural
change in the catalytic site, which turns on the kinase
activities. Recently, the LKB1 tumor suppressor kinase
[19] was reported to be a major upstream activator of

AMPK family kinases [20]. LKB1 phosphorylates Thr
residues in the A-loops of SIKs. By using LKB1-defect-
ive HeLa cells and a compound inhibiting TORC-
kinases, including SIKs, we tried to elucidate the
importance of the Ser133-independent activation of
CREB. The results suggested that the phospho ⁄ dephos-
pho regulation of TORC plays an indispensable role in
the regulated activation of CREB.
Results
All TORCs are substrates of SIK1
One of the downstream branches of the SIK-signaling
cascade leads to the regulation of CRE-dependent
Y. Katoh et al. Silencing of CREB by LKB1-SIK
FEBS Journal 273 (2006) 2730–2748 ª 2006 The Authors Journal compilation ª 2006 FEBS 2731
transcription, and we recently succeeded in identifying
TORC2, a CREB-specific coactivator, as an endog-
enous substrate of SIK2. Because mammals have three
TORC isoforms, we decided to clarify which isoform
could act as the endogenous substrate of SIK1.
Figure 1A shows that the SIK-phosphorylation motif
is highly conserved among the three isoforms. SIK1
was able to phosphorylate all the TORC peptides
except for the S171A-TORC2 (Fig. 1B).
The levels of SIK1-dependent phosphorylation of
TORC isoforms were also examined in cultured cells.
By using COS-7 cells, glutathione-S-transferase (GST)-
tagged full-length TORCs were coexpressed with con-
trasting SIK1 mutants; one was a kinase-defective
mutant (K56M) and the other was a mutant constitu-
tively phosphorylating TORC [17] (S577A mutant). As

shown in Fig. 1C, the levels of phosphorylation at
Ser171 of TORC2 and Ser163 of TORC3 were strik-
ingly elevated in the presence of SIK1 (S577A) (see the
lanes indicated by 577). However, the corresponding
residue, Ser167 of TORC1, seemed to be phosphor-
ylated even in cells expressing inactive SIK1 (the lanes
indicated by 56), and its phosphorylation level was
enhanced slightly in cells expressing SIK1 (S577A). In
contrast to the phosphorylation at Ser167, binding of
14-3-3 to TORC1 was significantly enhanced by SIK1
(S577A), suggesting that SIK1 could phosphorylate
TORC1, but some as yet unidentified kinases, other
than SIK1, might phosphorylate at Ser167.
To evaluate the direct action of SIK1 on the trans-
activation activity of TORCs, assays were performed
using Gal4-fused TORCs (Fig. 1D). SIK1 was able to
completely inhibit the transactivation activities derived
from all TORCs. Together, these results suggested that
SIK1 could phosphorylate all TORCs and thereby
repress their transactivation activities.
SIK1 is unable to induce the nuclear export
of TORC in HeLa cells
The nucleo-cytoplasmic redistribution of TORC2 is
important for both the stimuli-induced CRE activation
AB
DC
Fig. 1. SIK affects the functions of all TORCs. (A) Amino acid alignment of the SIK phosphorylation motif (box) of TORC1–3. SIK2 was
shown to phosphorylate Ser171 of TORC2 [14]. The corresponding Ser residues, Ser167 (TORC1), Ser171 (TORC2) and Ser163 (TORC3) are
indicated in bold. (B) GST–TORC peptides were prepared in E. coli and used as substrates for an in vitro kinase assay ([
32

P]dATP[aP]). GST-
tagged SIK1(1–354) prepared in COS-7 cells was used as an enzyme. (Upper) Incorporation of
32
P into GST–TORC peptides. (Lower)
Coomassie Brilliant Blue (CBB) staining of the substrates. GST–Syntide2 was used as a positive control substrate. (C) COS-7 cells were co-
transformed with pEBG-TORC1 (1.5 lg), pEBG-TORC2 (4 lg) or pEBG-TORC3 (6 lg) and pTarget-SIK1s (2 lg each). pEBG is a mammalian
expression plasmid for the GST-fusion protein. After 48 h, GST–TORCs were purified by glutathione columns (CP:) and then subjected to
western blot analyses (WB:) using anti-GST (upper), anti-(phospho-Ser171 TORC2) (middle) and anti-(14-3-3) (lower) sera. 577 indicates the
SIK1 mutant (S577A) which represses the CRE activity constitutively. 56 indicates the SIK1 mutant (K56M) with no kinase activity. (D)
HEK293 cells were cotransformed with the expression plasmids for Gal4-fusion TORC1–3 (0.05 lg) and a 5xGAL4-luciferase reporter plasmid
(0.2 lg) with an internal reporter phRL-TK(Int
–
) (0.03 lg) in the presence or absence of the SIK1 (S577A) plasmid (0.1 lg). The specific trans-
activation activities of TORCs were expressed as the fold-activation of the empty Gal4 vector, pM. Means and SD are indicated (n ¼ 4).
Silencing of CREB by LKB1-SIK Y. Katoh et al.
2732 FEBS Journal 273 (2006) 2730–2748 ª 2006 The Authors Journal compilation ª 2006 FEBS
and the SIK-mediated CRE repression. Interestingly,
Bittinger et al. found that TORC2 and TORC3 never
moved out of the nucleus in HeLa cells [15]. Because
HeLa cells lacked LKB1, which had been reported to
phosphorylate SIKs and activate them [20], we thought
that the impaired nuclear export of TORC2 in HeLa
cells was a result of the loss of SIK activity [20]. To
test this, we compared the behaviors of TORCs in
HeLa and COS-7 cells using a green fluorescent pro-
tein (GFP)-fusion technique. Because GFP–TORC1
was present in the cytoplasm of COS-7 cells (Fig. 2A),
we were unable to see the SIK1-induced intracellular
redistribution of GFP–TORC1 [compare SIK1(–) with
SIK1(+)]. In contrast to TORC1, GFP–TORC2

clearly showed SIK1-dependent nuclear export. GFP–
TORC3 also moved out of the nucleus in a slightly
lower level. As expected, overexpression of SIK1 did
not induce the intracellular redistribution of TORCs in
HeLa cells (Fig. 2B).
Overexpression of LKB1 in HeLa cells restores
the nucleo-cytoplasmic shuttling of TORC2
To elucidate the mechanism underlying the impaired
nucleo-cytoplasmic shuttling of TORC in HeLa cells,
we first tested whether overexpression of LKB1 in this
cell line could restore the SIK1-induced nuclear export
of TORC2 (Fig. 3A). As shown in the third panel of
the ‘upper’ set, a small population of GFP–TORC2
moved to the cytoplasm in LKB1-overexpressing HeLa
cells. Furthermore, expression of LKB1 and SIK1 in
combination could completely induce the nuclear
export of GFP–TORC2 (final panel). As expected, dis-
tribution of GFP–TORC2 was not influenced by the
overexpression of LKB1 in LKB1-positive COS-7 cells
(lower set).
The Thr182 of SIK1 is phosphorylated by LKB1,
resulting in conversion from inactive SIK1 to the act-
ive form. The importance of phospho-Thr182 was also
supported by the fact that substitution of the Thr with
a negatively charged residue produced a constitutive
active enzyme [20]; hence we prepared the T182E
mutant. As shown in Fig. 3B, however, neither SIK1
(T182E) nor SIK1 (T182A) could enhance LKB1-sup-
ported nuclear export of GFP–TORC2 in HeLa cells.
Differential properties of the A-loops of the

individual isoforms of SIK1–3
Because the SIK1 (T182E) mutant did not induce the
nuclear export of TORC2, we assayed the kinase activ-
ity of this mutant. The T182E mutant, prepared as a
GST-fusion protein using COS-7 cells, was much less
active than wild-type SIK1 (Fig. 4A). As expected,
A
B
Fig. 2. SIK1 alone is unable to induce the nuclear export TORC2 in
HeLa cells. (A) COS-7 cells cultured on cover slips were cotrans-
formed with expression vectors for GFP-tagged TORC1–3 with (+)
or without (–) the SIK1 (S577A) plasmid. After 24 h, the cells were
fixed for cytochemical analyses as described in Experimental proce-
dures. Green fluorescent signals of GFP–TORC1–3 (upper) and blue
fluorescent signals of nuclear staining with DAPI (lower) are shown.
(B) The same experiments were performed using HeLa cells. More
than 80% of GFP-positive cells had similar patterns as shown in
each representative panel.
A
B
Fig. 3. LKB1 is essential for the SIK1-induced nuclear export of
TORC2 in HeLa cells. (A) LKB1-defective HeLa cells (upper) and
LKB1-positive COS-7 cells (lower) were cotransformed with the
GFP–TORC2 expression plasmid and SIK1 expression plasmid in
the presence or absence of the LKB1 expression plasmid (pEBG-
LKB1). (B) Thr182, the LKB1-dependent phosphorylation site, of
SIK1 was substituted with Glu or Ala, and the resultant mutants
were subjected to cytochemical analyses of GFP–TORC2. HeLa
cells were transformed with plasmids as in Fig. 2.
Y. Katoh et al. Silencing of CREB by LKB1-SIK

FEBS Journal 273 (2006) 2730–2748 ª 2006 The Authors Journal compilation ª 2006 FEBS 2733
neither SIK1 (T182A) nor a negative control mutant,
K56M, showed kinase activities. The discrepancy
between our T182E mutant and the mutant in previous
reports [20,21] might be caused by the different
sources, Escherichia coli or cultured cells. However,
similar discrepancies have also arisen in studies of
AMPK [22].
There are three isoforms of SIK in the AMPK-rela-
ted kinase family. Although the overall sequence of
A-loops in SIKs is highly conserved (Fig. 4B), some
variety is found in the N-terminal side of the LKB1-
phosphorylatable Thr in SIK3. To see whether the
SIK2 and SIK3 isoforms behave similarly to SIK1
with regard to LKB1-dependent phosphorylation of
Thr, we prepared several mutants in which the corres-
ponding Thr residues were substituted. Kinase assays
of GST–SIK2s (Fig. 4C) produced results similar to
those of SIK1s. However, GST–SIK3s (Fig. 4D) provi-
ded results quite different from the others. SIK3
(T163A) had a little peptide phosphorylation activity,
and SIK3 (T163E) had activity as high as that of the
wild-type enzyme.
SIK kinase activity is sufficient to induce the
nuclear export of TORC2 in HeLa cells
The finding of the constitutive active SIK3 mutant,
SIK3 (T163E), prompted us to investigate whether the
kinase activity of SIK was sufficient to export TORC2
even under LKB1-defective conditions. Expression
plasmids for GFP–TORC2 and SIK3s were cotrans-

formed into HeLa cells. In LKB1-overexpressing HeLa
cells, GFP–TORC2 was exported from the nucleus to
the cytoplasm by either wild-type SIK3 or SIK3
(T163E) mutant (Fig. 5, upper). SIK3 (T163A) mutant
was unable to enhance the nuclear export of GFP–
TORC2. As expected, even in LKB1-nonexpressing
HeLa cells (Fig. 5, lower), SIK3 (T163E) could induce
the nuclear export of GFP–TORC2, although wild-
type SIK3, induced little export. These results suggested
A
B
DC
Fig. 4. The effect of the Thr to Glu substitu-
tion in the A-loop on the kinase activities of
SIKs. (A) GST–SIK1 (1–354) and its mutants
were prepared using COS-7 cells and were
subjected to an in vitro kinase assay. K56M
is kinase-defective SIK1, a negative control
mutant. GST–Syntide2 was used as a sub-
strate. (B) Alignment of amino acid
sequences of the A-loops of SIK1–3 and
AMPKa1. Conserved residues are marked
by black boxes, and the Thr residues repor-
ted to be phosphorylated by LKB1 are indi-
cated by a phosphor symbol. (C) Thr175 of
GST–SIK2 (full-length), corresponding to
Thr182 of SIK1, was replaced with Glu or
Gly, and the resultant mutants were subjec-
ted to an in vitro kinase assay. K49M is kin-
ase-defective SIK2. (D) Thr163 of the SIK3

kinase domain (1–340) was substituted with
Ala or Glu. K37M is kinase-defective SIK3.
These panels were one of representative
sets using SIK enzymes that had been pre-
pared using COS-7 cells at least three
times.
Fig. 5. The constitutive active SIK3 exports TORC2 without LKB1
in HeLa cells. The effects of substitutions at Thr163 of SIK3 on the
intracellular localization of GFP–TORC2 in HeLa cells in the pres-
ence (upper, +LKB1) or absence (lower, –LKB1) of LKB1 as des-
cribed in Figs 2 and 3. pEBG–SIK3 (1–340) was used for the
overexpression of SIK3.
Silencing of CREB by LKB1-SIK Y. Katoh et al.
2734 FEBS Journal 273 (2006) 2730–2748 ª 2006 The Authors Journal compilation ª 2006 FEBS
that LKB1 could regulate the intracellular distribution
of TORC2 through SIKs, and that SIK kinase activity
might be sufficient to induce the nuclear export of
TORC2.
Environments of CRE-dependent transcription
in HeLa cells
Next, we examined the expression of an endogenous
target of CREB, NR4A2 (Nurr1) gene (Fig. 6A). The
level of NR4A2 mRNA was significantly induced by
forskolin treatment in HEK293 cells. In HeLa cells,
however, it had already been expressed moderately,
and its level was not enhanced strongly by forskolin
treatment. The level of 36B4 RNA, generally used as
an internal standard, showed no change.
To find out why HeLa cells expressed NR4A2
mRNA constitutively, we compared the expression

level and the status of components in the TORC–
CREB system between HeLa and HEK293 cells. The
mRNA levels of TORCs in HeLa cells did not differ
substantially from those in HEK293 cells (Fig. 6B).
However, protein levels of TORCs seemed to be much
lower in HeLa cells than in HEK293 cells (Fig. 6C,D).
TORC2 proteins in HEK293 cells are known to
migrate as two bands on SDS⁄ PAGE (Fig. 6C).
A slowly moving form was the major form in nonstim-
ulated HEK293 cells and was shown to be the phos-
phorylated form. However, the rapidly moving form
was the major form in forskolin-treated cells and was
shown to be the dephosphorylated form [14,15].
In HeLa cells, however, only the rapidly moving
AB
DC
E
Fig. 6. Impaired CRE-dependent transcrip-
tion in HeLa cells. (A) Quantification of the
mRNA levels for NR4A2 and 36B4 in
HEK293 and HeLa cells using real-time PCR
analyses. Forskolin (20 l
M) was added to
the culture medium 4 h prior to the harvest
for total RNA extraction. One unit is equival-
ent to 6 pg of the standard plasmids con-
taining respective amplicons. To quantify
36B4 RNA, a reverse-transcribed mixture
was further diluted at 1:100. Means and SD
are indicated (n ¼ 3). (B) Quantification of

the mRNA levels for TORC1–3 in HEK293
and HeLa cells by real-time PCR analyses.
Forskolin (20 l
M) was added to the culture
medium 4 h prior to the harvest for total
RNA extraction. (C) Analyses of the level
and modification of the TORC2 protein in
HEK293 and HeLa cells. Forskolin (20 l
M)
was added to the culture medium 2 h prior
to the harvest for immunoprecipitation (IP)
followed by western blot analyses (WB). (D)
Analyses of the levels and modifications of
TORC1 and TORC3 in HEK293 and the
HeLa cells. (E) The phosphorylation of CREB
at Ser133 is impaired in the HeLa cells.
Forskolin (20 l
M) was added to the culture
medium 30 min prior to the harvest for
western blotting.
Y. Katoh et al. Silencing of CREB by LKB1-SIK
FEBS Journal 273 (2006) 2730–2748 ª 2006 The Authors Journal compilation ª 2006 FEBS 2735
dephospho form was seen. Because our antibody raised
against TORC1 was able to detect both TORC1 and
TORC3 with equal efficiency, analyses of these two
TORCs were carried in a single blot (Fig. 6D). Simi-
larly to the case of TORC2, TORC1 and TORC3 also
formed two bands in the gel. The cases of TORC1 and
TORC3 were apparently the same as TORC2, but
might be substantially different, because all of the

bands responded to forskolin treatment in HEK293
cells and shifted to lower positions. However, in HeLa
cells the bands remained at the same positions irres-
pective of treatment. It should be mentioned here that
Ser133 phosphorylation of CREB might not be strong
in forskolin-treated HeLa cells (Fig. 6E), suggesting
that more than one step in the regulatory pathway of
CREB might be impaired in HeLa cells.
LKB1 restores the accentual regulation of
CRE-dependent transcription
To examine whether the constitutive expression of
NR4A2 mRNA in HeLa cells suggested the impaired
regulation of CREs, and if so, to test whether LKB1
could restore the forskolin-induced activation of CRE-
dependent transcription, we tried to perform reporter
assays. Because plasmid-based reporters could not pro-
vide a high enough level of reporter activities in HeLa
cells (not shown), we prepared an adenovirus-mediated
reporter system. As shown in Fig. 7A, weak enhance-
ment of CRE activity by forskolin was observed in the
control HeLa cells (LacZ). Coinfection with the LKB1-
adenovirus (LKB1) repressed basal CRE activity to
one-tenth of its original level within 24 h. At this time
point, forskolin was unable to substantially induce
CRE activity. At 48–72 h post infection, however, a
large induction of the CRE activity by forskolin was
observed. In addition, forskolin induced NR4A2
mRNA in LKB1 expressing HeLa cells (Fig. 7B).
To investigate whether the impaired regulation of
CRE-dependent transcription in HeLa cells resulted

from the dysfunction of the overall phosphorylation
cascades in the SIK–TORC system or the particular
combination of SIK- and TORC isoforms, the levels of
proteins and the phosphorylation of individual isoforms
were examined. As shown in Fig. 7C, the level of SIK1
protein was elevated slightly by forskolin in control
cells (LacZ). When the LKB1-adenovirus was infected,
the basal level of SIK1 decreased significantly, and the
level was elevated prominently by forskolin. These
results agreed with the fact that SIK1 gene expression
depended on its own CREs [18]. In vitro kinase assays
using the SIK1 protein purified by immunoprecipitation
indicated that overexpression of LKB1 restored SIK1
kinase activity in HeLa cells (see the panel indicated by
32
P-ATP. As expected, Thr182 was not phosphory-
lated in the LKB1 nonexpressing HeLa cells. Because
sensitivity of the anti-(phospho-Thr182 IgG) was less
than that of the anti-(SIK1 IgG), we could not dis-
cuss whether Thr182 was phosphorylated in the LKB1-
expressing cells when cells were not stimulated with
forskolin (third lane from the left). After forskolin
treatment, however, the level of SIK1 protein had risen
sufficiently so that we were able to detect phospho-
Thr182 (the final lane).
In the case of SIK2 (Fig. 7D), the protein level was
not influenced by overexpression of LKB1. The levels
of kinase activity and phospho-Thr175 were elevated
in the LKB1-expressing HeLa cells. By contrast, the
protein level of SIK3 increased in LKB1-expressing

HeLa cells, and restoration of the kinase activity and
phosphorylation at Thr163 also occurred (Fig. 7E).
Next, we examined the phosphorylation of TORCs
in the same way (Fig. 7F). Similarly to the cases for
HEK293 cells, overexpression of LKB1 restored phos-
pho- ⁄ dephospho regulation of TORCs in HeLa cells.
We describe briefly here the results of TORC1. A small
part of the TORC1 population had already been phos-
phorylated in control HeLa cells (lanes indicated by
LacZ), and LKB1 enhanced the phosphorylation of
TORC1 (third lane from the left). Forskolin treatment
stimulated the dephosphorylation of TORC1 a little
(final lane). Results in Figs 7F and 1C suggest that
multiple cascades might be operating differentially in
the phosphorylation of TORC1, and these cascades
may be classified into three categories, LKB independ-
ent, SIK independent and SIK dependent.
Finally, we assayed the level of CREB phosphoryla-
tion (Fig. 7G). Forskolin-induced phosphorylation of
CREB at Ser133 was evident in LKB1-overexpressing
HeLa cells. These results suggested that LKB1 could
modulate the actions of all participants in the SIK–
TORC–CREB cascade.
SIK activity restores the regulation of
CRE-dependent transcription in HeLa cells
To obtain direct evidence of SIK-mediated phosphory-
lation of TORC in HeLa cells, the constitutive active
SIK3 mutant was overexpressed in this cell line. As
shown in Fig. 8A, the constitutive active SIK3 mutant,
T163E, could phosphorylate TORC2 even in the

absence of LKB1. Moreover, the T163E mutant could
recover the forskolin-dependent induction of CRE
activity without LKB1 (Fig. 8B).
Finally, it should be noted that the forskolin-
induced CREB phosphorylation at Ser133 was restored
Silencing of CREB by LKB1-SIK Y. Katoh et al.
2736 FEBS Journal 273 (2006) 2730–2748 ª 2006 The Authors Journal compilation ª 2006 FEBS
by overexpression of SIK3 (T163E) (Fig. 8C). These
observations suggested that restoration of SIK activity
might be sufficient for repairing impaired CREB-
dependent transcription in HeLa cells.
LKB1-mediated phosphorylation of SIK1 at
Thr182 enhances phosphorylation at Ser577
We noticed a large discrepancy between the increase in
total SIK1 activity and the decrease in the level of
TORC phosphorylation in forskolin-treated LKB1-
expressing HeLa cells (Fig. 7C,F). In this regard, we
found a similar case when CREB was activated by
PKA; PKA also phosphorylated SIK1 at Ser577,
which diminished SIK1-mediated cytoplasmic retention
of TORC2 [17]. Interestingly, phosphorylation at
Ser587 of SIK2 (corresponding to Ser577 of SIK1) was
not obvious in LKB1 nontransformed HeLa cells
(Fig. 7D, lower). To compare the specific level of SIK1
phosphorylation at Ser577 with that at Thr182 and the
AB
DE
C
FG
Fig. 7. LKB1 restores forskolin-induced CRE activity in HeLa cells. (A) HeLa cells were cotransfected with adenovirus reporters, Ad-CRE-fLuc

or Ad-TK-rLuc (an internal standard), at moi 3 and an LKB1-adenovirus or a lacZ-adenovirus at moi 30. After the indicated periods, cells were
harvested for the luciferase assays. Forskolin (20 l
M) was added to the culture medium 8 h prior to the harvest. (B) HeLa cells were trans-
fected with the adenovirus of LKB1 or lacZ. After 72 h, total RNA was purified from the cells and the levels of mRNA for NR4A2 (Nurr1) and
36B4 were quantified using real-time PCR analysis as described in the Experimental procedures. Forskolin (20 l
M) was added to the culture
medium 8 h prior to the harvest. n ¼ 3. (C–G) HeLa cells were transfected with the LKB1-adenovirus or the lacZ-adenovirus. After 72 h, cells
were treated with forskolin (20 l
M) for 1 h and then subjected to immunoprecipitation (IP:) using anti-SIK1 (C), anti-SIK2 (D), anti-SIK3 (E),
anti-TORC2 or anti-TORC1 ⁄ 3 (F) sera followed by western blotting. Immunopurified SIK enzymes were also subjected to in vitro kinase
assays ([
32
P]dATP[aP]) using GST–Syntide2 as a substrate. The panels represent one of duplicate experiments. Anti-(phospho-Thr182 SIK1)
was able to detect phospho-Thr175 of SIK2 and phospho-Thr163 of SIK3. Anti-(phospho-Ser577 SIK1) cross-reacted with phospho-Ser587 of
SIK2. To detect overexpressed LKB1 and total- ⁄ phospho-CREB, cell lysate was subjected to western blotting (G).
Y. Katoh et al. Silencing of CREB by LKB1-SIK
FEBS Journal 273 (2006) 2730–2748 ª 2006 The Authors Journal compilation ª 2006 FEBS 2737
kinase activity, GST-fusion SIK1 was overexpressed
in HeLa cells. As shown in Fig. 9A, Ser577 was
not phosphorylated in control HeLa cells and was
apparently less phosphorylated by forskolin treatment
(indicated by LacZ). Overexpression of LKB1 induced
phosphorylation at Ser577, and its level was signifi-
cantly elevated after forskolin treatment (indicated by
LKB1), suggesting that phospho-Ser577 might be the
result of an autophosphorylation of SIK1. Other indi-
cators, such as phospho-Thr182 and kinase activities,
depended on LKB1, but not on the forskolin treat-
ment.
When Ser577 is phosphorylated, the phospho-SIK1

moves to the cytoplasm. Using this property, we exam-
ined the LKB1-initiated autophosphorylation of SIK1
at Ser577. As shown in Fig. 9B, in control HeLa cells
(–), GFP–SIK1 was localized only in the nucleus.
When GFP–SIK1 was coexpressed with LKB1 or
PKA, part of the SIK1 population moved to the cyto-
plasm. LKB1-induced nuclear export of SIK1 was
abolished by the T182A substitution (Fig. 9C). Substi-
tution at Ser577 completely inhibited both LKB1- and
PKA-induced nuclear export of SIK1.
Finally, we tested the level of TORC phosphoryla-
tion using wild-type and S577A-SIK1 (Fig. 9D). In
COS-7 cells, the Ser577 mutant SIK1 phosphorylated
GST-fusion TORC2 more efficiently than the wild-
type. These observations suggested that the PKA-phos-
phorylatable Ser577 also acted as the autophosphory-
lation site, and that phospho-Ser577 might be a critical
modulator of the TORC phosphorylation activity of
SIK1. In this context, LKB1 might also play important
roles in the attenuation step of the phosphorylation of
TORC. The SIK3 T163E-mutant having the additional
mutation at Ser493, equivalent to S577A of SIK1, also
suggested the importance of the Ser phosphorylation
(Fig. 8B).
Inhibition of kinase cascades activates
CRE-dependent transcription constitutively
The constitutive activation of CRE-dependent tran-
scription in HeLa cells (Fig. 6A,B) might be due to
inactivation of the phosphorylation cascades from
LKB1 to TORCs, suggesting, paradoxically, that inhi-

bition of the kinase cascades could mimic impaired
CREB regulation even in LKB1-positive cells. To test
this possibility, we performed CRE-reporter assays in
HEK293 cells in the presence of various kinase inhibi-
tors. As shown in Fig. 10A, no specific kinase inhibitor
could activate the CRE. However, staurosporine (STS;
10 nm), a nonspecific kinase inhibitor [23], induced
CRE reporter activity to levels as high as forskolin.
Moreover, staurosporine upregulated transcription of
the NR4A2 gene to a level higher than that elevated by
forskolin treatment (Fig. 10B).
A
B
C
Fig. 8. The constitutive active SIK3 restores forskolin-induced CRE
activity without LKB1 in HeLa cells. (A) HeLa cells were infected
with the SIK3 (full-length) adenoviruses. After 72 h incubation,
TORC2 was analyzed by immunoprecipitation, and SIK3 and LKB1
were detected by western blotting using cell lysates. The lower
band in the SIK3 panel might be degraded products. (B) HeLa cells
were cotransformed with SIK3-adenoviruses and reporter-adeno-
viruses, and CRE activity was measured as described in Fig. 1.
S493A of SIK3 is equivalent to S577A of SIK1. (C) HeLa cells were
infected with the SIK3 T163E adenovirus or its control virus, LacZ.
After 72 h incubation, cells were harvested to analyze the level of
phospho-CREB using western blotting. These panels represent
experiments performed at least in duplicate.
Silencing of CREB by LKB1-SIK Y. Katoh et al.
2738 FEBS Journal 273 (2006) 2730–2748 ª 2006 The Authors Journal compilation ª 2006 FEBS
Staurosporine has been classified as a PKC inhibitor

(Fig. S1A). However, another PKC-specific inhibitor,
bisindolylmaleimide I (Bis) did not induce any CRE
reporter activity in our assay system (Fig. 10A), sug-
gesting that PKC might not be the kinase responsible
for staurosporine-induced CRE activity.
To investigate the phosphorylation status of TORC
and CREB in staurosporine-treated cells, GST-tagged
TORC2 and endogenous CREB were examined in
COS-7 cells (Fig. 10C). Forskolin induced both the
dephosphorylation at Ser171 and the decrease in the
level of bound 14-3-3. It enhanced the phosphoryla-
tion of CREB at Ser133, of course. As expected, sta-
urosporine completely inhibited the phosphorylation
of TORC2 and did not enhance the phosphorylation
of CREB.
Because staurosporine significantly blocked SIK1-
mediated CRE repression (not shown), the efficacy of
staurosporine on SIK1 was estimated by measuring its
IC
50
as regards the kinase activity. The in vitro IC
50
was  0.15 nm (Fig. S1B). To evaluate SIK1 inhibition
in vivo, the difference between forskolin-induced CRE-
reporter activity and its activity in the presence of
SIK1 S577A was used. An in vivo IC
50
of staurospo-
rine against the exogenously expressed SIK1 S577A
mutant was  5.0 nm (Fig. S1C). These results sugges-

ted that the kinase activity of endogenous SIK might
be inhibited by staurosporine at a dose as low as that
against PKC.
To examine whether staurosporine-induced dephos-
phorylation of TORC2 was accompanied by its nuclear
accumulation, GFP–TORC2 was expressed in HeLa
cells in the presence or absence of the LKB1–SIK cas-
cades, and the cells were treated with staurosporine
(Fig. 10D). Staurosporine inhibited the nuclear export
of GFP–TORC2 in all the cases tested. These results,
however, might indicate two possibilities, namely that
staurosporine either inhibited SIKs directly or blocked
the upstream cascades of SIKs, including LKB1.
To clarify this point, COS-7 cells that had been
expressing GST-tagged SIK1 were treated with sta-
urosporine and GST–SIK1 protein was purified
(Fig. 10E). SIK1 enzyme purified from the staurospo-
rine-treated cells was phosphorylated at Thr182 but
did not show any kinase activities. Because Thr172 of
AMPKa1 (corresponding to Thr182 of SIK1) is also
phosphorylated by LKB1, we performed the same
experiment using GST–AMPKa1 (lower left). Neither
the phosphorylation level at Thr172 nor the kinase
activity of AMPKa1 was affected by staurosporine
treatment. Forskolin treatment did not alter the levels
of Thr phosphorylation or the kinase activity of SIK1
AB
C
D
Fig. 9. Ser577 is an autophosphorylation site of SIK1. (A) HeLa cells were transformed with the expression plasmid, pEBG-SIK1 for GST-

fusion SIK1 (full-length), then transfected with the lacZ- or LKB1-adenovirus. After 48 h incubation, the cells were treated with forskolin
(20 l
M) for 30 min, and the GST–SIK1 protein was purified using a glutathione column (CP). The SIK1 protein was subjected to western blot
analyses as well as kinase assays as described in Fig. 7. (B) HeLa cells were transformed with the GFP–SIK1 expression plasmid with the
LKB1- or the PKA-expression plasmid as described in Fig. 2. (C) Mutant GFP–SIK1, T182A or S577A, was expressed in LKB1-expressing
HeLa cells with or without PKA. (D) COS-7 cells were transformed with the GST–TORC2 expression plasmid in the presence of the SIK1
expression plasmid, wild-type or S577A. After 48 h incubation, the cells were treated with forskolin (20 l
M) for 1 h, and then the GST–
TORC2 protein was purified.
Y. Katoh et al. Silencing of CREB by LKB1-SIK
FEBS Journal 273 (2006) 2730–2748 ª 2006 The Authors Journal compilation ª 2006 FEBS 2739
and AMPKa1. Similar experiments using other SIKs
were also performed (right). Staurosporine inhibited
the kinase activity of SIK2 and SIK3 in cultured cells.
These results suggested that staurosporine inhibited the
kinase activities of SIKs directly without blocking the
upstream cascades.
Inhibition of SIK may be a direct cause of
staurosporine-induced CREB activation
If the major cause of the staurosporine-induced dep-
hosphorylation of TORC is the direct inhibition of
SIKs, CRE activity may be re-repressed by introducing
ABC
E
D
Fig. 10. Inhibition of kinase cascades including SIKs constitutively activates CRE-dependent transcription. (A) The screening of protein kinase
inhibitors having a potency to induce CRE-dependent transcription. HEK293 cells that had been transformed with the pTAL-CRE reporter
(f Luc) and an internal standard reporter, pRL-SV40 (r Luc), were treated with forskolin (20 l
M), H89 (1 lM), KN93 (10 lM), bisindolylmalei-
mide I (Bis: 20 l

M), PD98059 (PD: 20 lM), Wortmannin (Wort: 1 lM), SB203580 (SB: 20 lM), Hypericin (Hyper: 2 lM), KT5823 (20 lM), sta-
urosporine (STS: 10 n
M), or only DMSO (0.1%) for 12 h. Means and SD are indicated (n ¼ 4). (B) HEK293 cells were treated with forskolin
(20 l
M) or staurosporine (STS: 10 nM) for 4 h and then harvested to quantify the levels of NR4A2 and 36B4 RNA. n ¼ 3. (C) COS-7 cells that
had been transformed with pEBG-TORC2 were treated with forskolin (20 l
M) or staurosporine (STS: 10 nM) for 2 h. GST–TORC2 was puri-
fied using a glutathione column and the phosphorylation level of TORC2 was examined by western blotting using anti-(phospho-Ser171) and
anti-(14-3-3) sera. The nuclear lysates were used to detect CREB. (D) HeLa cells that had been overexpressing GFP–TORC2 with SIK1–3 in
the presence of LKB1 were treated without or with staurosporine (10 n
M) for 1 h (E) COS-7 cells that had been expressing GST–SIK1 (1–
354) or GST–AMPKa1(2–312) were treated with forskolin (20 l
M) or staurosporine (10 nM) for 2 h. Purified GST–SIK1 and GST–AMPKa1
were subjected to western blot analyses or an in vitro kinase assay (left sets). The same experiments without forskolin treatment were car-
ried out using SIK2 (1–348) and SIK3 (1–340) (right sets). The GST-p300 peptide (LLRSGSSPNL) was used as the substrate, because
AMPKa1 phosphorylated the GST–Syntide2 peptide a little.
Silencing of CREB by LKB1-SIK Y. Katoh et al.
2740 FEBS Journal 273 (2006) 2730–2748 ª 2006 The Authors Journal compilation ª 2006 FEBS
a staurosporine-resistant mutation into SIK. A model
of staurosporine accommodated in the ATP-binding
pockets of SIKs suggested possible interactions
between three oxygen atoms, each derived from three
glutamic acid residues, and the nitrogen atoms of sta-
urosporine. Two of the three oxygens were constituents
of peptide bonds, whereas the third was in a gamma-
carboxyl group. We focused our attention on the
glutamic acid residue whose gamma-carboxyl group
interacted with staurosporine, and decided to replace it
with an aspartic acid residue (Fig. 11A shows the case
of SIK2). Thus mutants SIK1 (E110D), SIK2 (E103D)

and SIK3 (E91D) were prepared. The kinase activity
of SIK1 (E110) and SIK3 (E91) was lower than those
of their parents (not shown), and therefore these
mutants did not seem suitable for the analysis of sta-
urosporine action. Mutant SIK2 (E103D), however,
showed kinase activity comparable with the parent and
the activity was not influenced by the addition of
staurosporine in vitro (Fig. 11B). SIK2 (E103D) in the
reporter assay systems repressed forskolin-induced
CRE activity as strongly as the wild-type (Fig. 11C,
left). When staurosporine was added to this assay sys-
tem, the SIK2 (E103D)-dependent repression was less
influenced than the wild-type (right). These results sug-
gested that staurosporine may inhibit SIK directly,
which might result in the dephosphorylation of TORC
followed by the constitutive activation of CRE-
dependent transcription.
AMPK might not regulate the CREB activity
directly
Recently, Shaw et al. [24] showed that LKB1 downreg-
ulated the expression of gluconeogenic genes in the
liver, possibly via activation of AMPK. Koo et al. [18]
also showed that AMPK possibly phosphorylated
TORC2 and blocked its nuclear accumulation in the
liver. However, the degree to which an AMPK agonist
enhanced the phosphorylation of TORC2 was lower
A
B
C
Fig. 11. Staurosporine-resistant SIK2 repres-

ses staurosporine-induced CRE activity. (A)
Model of the ATP-binding pocket of SIK2.
The model of the staurosporine–SIK2 com-
plex was prepared as described in Experi-
mental procedures. Three oxygen atoms in
glutamic acids, E97, E103 and E146, poss-
ibly interact with nitrogen atoms of sta-
urosporine (dotted lines in the left-hand
panel). To disrupt the interaction between
the ATP-binding pocket and staurosporine
by amino acid substitution, E103 was chan-
ged to Asp (right). (B) The GST–SIK2 pro-
tein, wild-type or E103D, was prepared in
COS-7 cells and subjected to the in vitro kin-
ase assay in the presence of staurosporine
at the indicated concentrations. The GST–
TORC2 peptide was used as the substrate.
(C) HEK293 cells that had been transformed
with 0.2 lg of the CRE-luciferase reporter
(pTAL-CRE) or its empty vector (pTAL) and
an internal standard reporter (pRL-SV40:
0.03 lg) in the presence or absence of the
SIK2 (wild-type or E103D) expression plas-
mid (indicated amount) were treated without
or with staurosporine (10 n
M) in the pres-
ence or absence of forskolin (20 l
M) for 8 h.
To minimize the amount of the SIK2 protein,
it was expressed as S587A mutants. n ¼ 2.

Y. Katoh et al. Silencing of CREB by LKB1-SIK
FEBS Journal 273 (2006) 2730–2748 ª 2006 The Authors Journal compilation ª 2006 FEBS 2741
than that by overexpressed SIKs. Moreover, it had not
been shown whether CRE was the element responsible
for the AMPK agonists-induced downregulation of
gluconeogenic gene expression. Finally, a low dose of
staurosporine (10 nm) could induce CRE-dependent
transcription without significant effects on AMPK
in living cells (Fig. 10E; also see in vitro IC
50
in
Fig. S1B). To examine the potential of AMPK-related
kinases for CREB-regulatory activity, we performed
reporter assays using a CRE reporter and a Gal4–
TORC2 reporter. As shown in Fig. 12A, overexpres-
sion of the kinase domains (amino acids 1–312) of
AMPKa1 and -a2 did not repress the reporter activit-
ies derived from the CRE reporter plasmid. This was
also the case when Gal4–TORC2 was used in the
reporter system (Fig. 12B). All enzymes were expressed
as active kinases in COS-7 cells and could phosphory-
late the TORC2 peptide in vitro (Fig. 12C).
Because the signal transduction of AMPKa is accel-
erated by the interaction with b and c subunits, the
full-length AMPKa subunit was expressed in the CRE
reporter assay in the presence or absence of b and c
subunits. As shown in Fig. 12D, no combination of
AMPKas ⁄ b1 ⁄ c1 had a significant effect on CRE activ-
ity. Exogenously expressed AMPKa may be active, as
it was highly phosphorylated at Thr172 (Fig. 12E) [25].

Moreover, although endogenous AMPKa may have a
molecular mass of  65 kDa, anti-(phospho-Thr172
IgG) did not react with bands around 65 kDa, suggest-
ing that the level of the exogenously expressed AMPK
might be much higher than that of endogenous
enzyme in our system. These results suggest that the
potential of AMPK as the regulator of CRE-depend-
ent transcription might be lower than those of SIKs in
living cells.
Finally, we would like to briefly mention MARK4.
Because the kinase domains of SIKs are highly con-
served with those of four other kinases that belong to
the MARK subfamily, we tested the possibility of
MARKs as CREB regulators by using one MARK,
MARK4. The kinase domain of MARK4 also showed
CREB-repression activity. In addition, the kinase
activity of the GST-fusion MARK4 was inhibited by
10 nm staurosporine in living COS-7 cells (not
shown).
Discussion
LKB1 is the kinase responsible for causing Peutz–Jeg-
hers syndrome [19] and has been reported to activate
AMPK-related kinases by phosphorylating Thr resi-
dues in the A-loops [20]. Because patients with Peutz–
Jeghers syndrome are at a high risk of developing
cancers of epithelial tissue origin, LKB1 has been sug-
gested as a tumor suppressor kinase [26].
SIK1, a member of the AMPK family, was shown
to be a downstream kinase of LKB1, with its Thr182
being phosphorylated by LKB1 [20]. Meanwhile, SIK,

when overexpressed in cells, was shown to inhibit
CREB activity by phosphorylating, and repressing,
CREB-specific coactivators, TORCs. TORC1 has been
independently identified as a candidate for inducers of
salivary gland tumors and it has been called mucoepi-
dermoid carcinoma translocated 1 (MECT1) [27]. The
genomic rearrangement of t(11;19), which is often
associated with mucoepidermoid carcinoma, produces
a fusion protein that has the N-terminal CREB bind-
ing region (1–42 amino acids) of TORC1 ⁄ MECT1
and the transcriptional activation domain of other
transcription factor, Mastermind-like gene family 2
(MAML2). The resultant chimeric protein, MECT1–
MAML2, binds to CREB, activates CRE-mediated
transcription [12] and induces foci formation in RK3E
cells [28]. Because the MECT1–MAML2 fusion protein
lacks Ser167 of TORC1 ⁄ MECT1, the SIK phosphory-
lation site (Fig. 1A), we speculate that loss of the SIK-
mediated repression of the MECT1–MAML2 activity
might cause the uncontrolled induction of CRE-
dependent transcriptions, which might result in tumor
formation. This is also supported indirectly by a recent
report that CREB has carcinogenic potential, when its
cellular level is kept high [29].
The question regarding whether the constitutive acti-
vation of CREB is one of the causes of Peutz–Jeghers
syndrome remains. Loss of one allele of the LKB1
gene is sufficient to induce Peutz–Jeghers syndrome
[30–32], which enhances the frequency of polyp forma-
tion. However, the polyps in Peutz–Jeghers syndrome

show resistance to transformation, and the progression
of carcinogenesis requires complete loss of the LKB1
gene [33]. However, neither knockdown of the LKB1
protein, using an siRNA technique, nor overexpression
of LKB1 affected CREB activity in HEK293 cells (not
shown). This was also supported by the result that
overexpression of LKB1 in COS cells did not alter the
intracellular distribution of GFP–TORC2 (Fig. 3A).
Therefore, we suppose that the constitutive activation
of CREB seen in HeLa cells may be due to complete
loss of the LKB1 protein, and, thus, the impaired
CREB regulation is unable to account for the patho-
genesis of Peutz–Jeghers syndrome caused by the
haploinsufficiency of the LKB1 allele.
Recent studies [20,34] have suggested similar proper-
ties for the A-loops of SIK1 and SIK3 (also called by
Qsk). Levels of kinase activities of SIK1 (T182E) and
SIK3 (T163E) were indistinguishable from those of
Silencing of CREB by LKB1-SIK Y. Katoh et al.
2742 FEBS Journal 273 (2006) 2730–2748 ª 2006 The Authors Journal compilation ª 2006 FEBS
wild-type enzymes phosphorylated by LKB1 [20,21].
A phospho-amino acid binding protein, 14-3-3, was
shown to associate with phospho-Thr in the A-loop of
SIK1 and SIK3, which resulted in enhancing their
kinase activities [34]. Association of 14-3-3 with the
A-loop of SIK2 (QIK) was reported as uncertain. At
AB
C
D
E

Fig. 12. SIKs and MARK4 have the potential to downregulate CRE-dependent transcription. (A) COS-7 cells were transformed with the CRE-
reporter plasmid (pTAL-CRE: 0.2 lg) in the presence or absence of the PKA expression plasmid (pIRES-PKA: 0.1 lg) and expression plas-
mids for the kinase domains of SIKs and related enzymes [pEBG: 0.2 lg; in addition to enzymes shown in Fig. 9E, MARK4 (1–380) and
AMPKa2 (1–312) were used]. After 24 h incubation, cells were harvested for a reporter assay. The CRE-dependent reporter activities that
had been normalized with internal reporter activities were expressed as fold of activities of the empty reporter (pTAL). (B) COS-7 cells were
transformed with the Gal4–TORC2 expression plasmid, the 5xGAL4-luciferase reporter plasmid and plasmids for the kinase domains of SIKs
and related enzymes (pEBG: 0.2 lg). Means and SD are indicated (n ¼ 4). (C) The expression level (upper) and the TORC2-peptide phos-
phorylation activity (lower) of the individual kinases were examined. The method for the in vitro kinase assay using antipTORC2 (pS171) IgG
was described previously [14]. (D) COS-7 cells were transformed with the CRE-reporter plasmid in the presence or absence of the PKA
expression plasmid and expression plasmids for the full-length AMPKa subunits with or without the b ⁄ c subunits. The AMPKa subunits
were expressed as GST-fusion proteins, and the b ⁄ c subunits were HA-tagged proteins. (E) Levels of expressed AMPKa and b ⁄ c subunits in
the cell lysate were examined by western blot analysis. The b ⁄ c subunits were detected by an anti-(HA-tag IgG). The active AMPKa subunits
were monitored by antiphospho-T172 IgG.
Y. Katoh et al. Silencing of CREB by LKB1-SIK
FEBS Journal 273 (2006) 2730–2748 ª 2006 The Authors Journal compilation ª 2006 FEBS 2743
least under our assay conditions, however, SIK3
(T163E), but neither SIK1 (T182E) nor SIK2 (T175E),
showed kinase activity as high as its wild-type enzyme
(Fig. 4). It remains to be examined whether 14-3-3
could bind to the glutamic acid of SIK3 (T163E), but
not to those of SIK1 (T182E) or SIK2 (T175E),
although to our knowledge no report has shown gluta-
mic acids acting as acceptors for 14-3-3. In addition,
Al-Hakim et al. suggested that the binding of 14-3-3 to
the A-loop of SIK1 might induce the cytoplasmic
localization of SIK1. However, as we show here, phos-
phorylation at Ser577, which occurred either by the
action of PKA or by autophosphorylation, might
cause the cytoplasmic localization of SIK1 (Fig. 9B,C).
In this context, phosphorylation at Thr182 in the

A-loop might be a prerequisite step for the cytoplasmic
localization of SIK1.
To summarize, although CREB is believed to be act-
ive when it is phosphorylated by kinases cascades, this
study gives a new insight that CREB has a constitutive
active potency, which is also repressed by kinase cas-
cades involving LKB1 and SIKs. In this study, how-
ever, to restore the regulated activation of CREB, we
used overexpression systems, which might not reflect
physiological levels. To elucidate the contribution of
individual TORC isoforms and TORC kinases, inclu-
ding SIKs, to CRE-dependent transcription in living
cells, it would be helpful to develop strategies using
the combination of RNAi. But when the total number
of the TORC kinases, their cross-talks and feedback
regulations are considered, these strategies would
require a quite formidable effort. At present, therefore,
our strategy to use staurosporine or LKB1-defective
cells is the only practical method to extract the consti-
tutive active potency of CREB without classical agon-
ists. Further elucidation of individual isoforms of SIK
and TORC will be needed.
Experimental procedures
Cells, chemicals and antibodies
HeLa S9 cells were purchased from the Health Sciences
Research Resource Bank (Osaka, Japan) and used as HeLa
cells in this study. Because the round shape of HeLa S9
cells was not suitable for cytochemical analyses, flat cells
were re-screened and used for the cytochemistry. 293A cells
were from Invitrogen (Carlsbad, CA) and used as HEK293

cells in this study. COS-7 cells were in a collection of our
laboratory. We used HEK293 cells and COS-7 cells as
LKB1-positive cells. Both originated from kidney fibro-
blasts and showed a good response to forskolin; therefore,
we used these cells for reporter assays. Because HEK293
cells are human cells, we used this cell line for mRNA ana-
lyses using real-time PCR. Because COS-7 cells have an
SV40-ori which amplifies plasmids episomally, we used this
cell line for the overexpression of SIKs or TORC2. In addi-
tion, HEK293 cells barely attached onto cover slips. There-
fore, we used COS-7 cells for cytochemical analyses.
4¢,6-Diamidino-2-phenylindole (DAPI) dilactate was
purchased from Molecular Probes (Eugene, OR), staurospo-
rine, H89, KN93, bisindolylmaleimide, PD98059, Wortman-
nin, SB203580, KT5823 and Hypericin were from
Calbiochem (San Diego, CA) and forskolin was from
Sigma-Aldrich (St. Louis, MO). Anti-CREB IgGs were from
Cell Signaling Technology (Beverly, MA). An anti-(LKB1
IgG) was from Santa Cruz (Santa Cruz, CA). An anti-hem-
agglutinin (HA)-tagged IgG was from Roche (Indianapolis,
IN). An anti-(phospho-AMPKa) serum (pT172) was pur-
chased from Cell Signaling (Danvers, MA).
An anti-(SIK1 IgG) was purified using the SIK1 pep-
tide (499–776). An anti-SIK3 serum was raised against a
human SIK3 peptide (1106–1263). A TORC2 antiserum
was raised against the mouse TORC2 peptide (563–699),
and the specific IgG was purified using the same peptide.
TORC1- and TORC3-antisera were raised against human
TORC1 peptide (551–650) and TORC3 peptide (480–619),
respectively. Because these antisera cross-reacted with both

TORC1 and TORC3, the IgG was purified from the anti-
TORC1 serum using the TORC3 peptide. The resultant
anti-(TORC1 ⁄ 3 IgG) could recognize both TORC1 and
TORC3 with equal efficiency. The above peptides were pre-
pared as GST-fusion peptides in E. coli using a pGEX-6P3
expression vector and cleaved from GST by PreScission
Protease (Amersham Biosciences). A phospho-Thr182 SIK1
antiserum was raised against a keyhole limpet hemocyanin-
conjugated peptide of KSGELLApTWCGSPPY (human
SIK2 A-loop; pT means phospho-Thr). The specific IgG
was purified with an affinity column immobilized with a
peptide of KPGEPLSpTWCGSPPY (SIK1 A-loop). The
phospho-Thr182 SIK2 cross-reacted with all phospho-Thr
residues of SIK1-3. Supplementary material (Fig. S1D)
shows the specificity of the antiphospho-T182 IgG. The
anti-(phospho-SIK1) (pSer577) serum was as described in
Takemori et al. [16], and the anti-(phospho-TORC2)
(pSer171) serum was as in Screaton et al. [14].
Plasmids
The cDNAs for rat AMPKs have been described previously
[35]. Human SIK3 and MARK4 cDNAs (KIAA0999 and
KIAA1860, respectively) were gifts from Kazusa DNA
Institute (Chiba, Japan). The KIAA0999 clone had an extra
5¢-UTR that might come from a 5¢-upstream gene [human
ankyrin repeat and MYBD domain containing 1 mRNA
(Accession no. BC033495)]. Because this 5¢-UTR was not
found in a mouse SIK3 mRNA (Accession no.
Silencing of CREB by LKB1-SIK Y. Katoh et al.
2744 FEBS Journal 273 (2006) 2730–2748 ª 2006 The Authors Journal compilation ª 2006 FEBS
NP_081774), we supposed that the initial methionine codon

(ATG) might be at 438–440 of the KIAA0999 clone (Acces-
sion no. BAA76843). As the result, SIK3 is likely to con-
tain 1263 amino acids.
The mammalian expression vector, pCMVsport6, con-
taining full-length hTORC1 (IMAGE: 4938995), mTORC2
(IMAGE: 5345301) and hTORC3 (IMAGE: 6470060)
cDNAs, were purchased from Invitrogen. Site-directed mut-
agenesis was carried out using a kit, Quick change muta-
genesis (Stratagene) to create restriction sites. The resultant
cDNAs were digested with BamHI, BglII, or SpeI and NotI
(in the multicloning site), and the TORCs cDNA fragments
were ligated into the BamHI–NotI or the SalI–NotI site of
a GFP expression vector pEGFP-C for cytochemical stud-
ies, a pENTR-1A plasmid (Invitrogen) for adenovirus pre-
paration and a pM vector (Gal4-fusion) for reporter assays.
Mouse LKB1 cDNA fragment was amplified from total
RNA of mouse adrenal tumor Y1 cells by RT-PCR using
primers based on the mouse LKB1 cDNA sequence (Acces-
sion no. AF145287). The sequences of the primers were
5¢-TTTACTAGTATGGACGTGGCGGACCCCGAG and
5¢-TTTGCGGCCGCTCACTGCTGCTTGCAGGCCGAG.
To prepare adenovirus vectors, cDNA fragments and
reporter DNA(s) in the pENTR-1A vector were transferred
onto pAd ⁄ CMV ⁄ V5-DEST or pAd ⁄ PL ⁄ V5-DEST adenovi-
rus-DNA using the Gateway system (Invitrogen). pAd-lacZ
was used as a standard to determine the titers of individual
viruses. When HeLa cells were infected with adenovirus at
> 50 moi (multiplicity of infection for HEK293 cells), some
of cells died within 72 h. Therefore, the total moi of the
adenovirus was adjusted to 30 plus 6 for reporters (three

for the CRE-fLuc adenovirus and three for the internal
standard TK-rLuc adenovirus).
Immunoprecipitation, in vitro kinase assay,
reporter assay, cytochemical analysis and
quantitative PCR analysis (real-time PCR)
Immunoprecipitation and in vitro kinase assay was per-
formed as described previously [10]. The methods for repor-
ter assays have been described previously [11]. To introduce
plasmids into the cells, we used LipofectAMINE 2000
(Invitrogen) ⁄ Escort V (Sigma-Aldrich) mixture (1:1) was
used in this study. Luciferase activities were measured by
using the Dual-Luciferase Reporter Assay System (Prome-
ga, Madison, WI). For the CRE-reporter assay, HEK293
and HeLa cells (2 · 10
4
per well) were transformed with
SIKs expression plasmids (pIRES, pEBG, pTarget or
pIRES: 0.1–0.2 lg), the CRE-luciferase reporter [pTAL-
CRE or its empty reporter (pTAL): 0.2 lg], the PKA
expression plasmid (pIRES-PKA: 0.1 lg) and 0.03 lgofan
internal standard Renilla luciferase vector [pRL-SV40 for
standard assays or phRL-TK(Int
–
) for assays with TORC
expression plasmids]. To measure the transcriptional activit-
ies of TORCs, HEK293 cells were transformed with Gal4
DNA binding domain-linked TORC expression vectors
(pM-TORCs: 0.05 lg) and the 5 · GAL4-luciferase repor-
ter plasmid (pTAL-5 · GAL4: 0.2 lg).
For fluorescent cytochemical analyses, cells were cultured

on poly L-lysine coated coverslips (18-mm) (Matsunami
Co. LTD, Tokyo, Japan) using a 12-well dish. COS-7 and
HeLa cells (< 1 · 10
4
) were transformed with 0.2–0.5 lg
of expression vectors for GFP-TORCs in the presence or
absence of pEBG-SIKs or -LKB1 (0.3 lg). After 16 h,
the cells were fixed with 1 mL of 4% paraformaldehyde
dissolved in NaCl ⁄ Pi for 15 min, stained with DAPI
(1 ng mL
)1
in 0.01% Triton X-100 ⁄ NaCl ⁄ P
i
) for 5 min,
and then washed with NaCl ⁄ Pi four times. The cells on the
coverslip were embedded onto a slide glass using 50% gly-
cerol. On average, about 80% of cells showed detectable
GFP-TORCs signals in independent duplicate experiments.
Total RNA was extracted using the RNeasy kit (Qiagen,
Valencia, CA). cDNAs were prepared by reverse transcrip-
tion (RT) from one microgram total RNA using Super-
script III and random primers (Invitrogen). One-hundredth
of the RT products and standard plasmids were subjected
to real-time PCR analyses using the IQ SYBR Green
Supermix (Bio-Rad). The PCR program included 10 min of
denaturation at 95 °C and then 40 cycles at 95 °C for 15 s,
58 °C for 30 s and 72 °C for 30 s.
The specific primers were used (TORC1-F: 5¢-CACCTG
GCTCCTCTCCACA; and TORC1-R: 5¢-AGCTGCTGCT
CCAGAGACA, TORC2-F: 5¢-TGACCTCACCAACCTG

CACT; and TORC2-R: 5¢-GTGAGTCATGGTGTGGGT
CA, TORC3-F: 5¢-CAACATCCCAGCTGCTATGA; and
TORC3-R: 5¢-GATGCGTTGGGAACAGATGT, NR4A2-
F: 5¢-ACCAGAACTACGTGGCCACTA; and NR4A2-R:
5¢-TCGAAGCGCATCTGGCAACTA, 36B4-F: 5¢-TGTG
TGTCTGCAGATCGGGT; and 36B4-R: 5¢-TGGATCAG
CCAGGAAGGCCT).
Modeling of a three-dimensional structure
of SIK1 with staurosporine
The three-dimensional structure of the SIK2 kinase domain
was modeled from the primary sequence of the rat SIK2
kinase domain using SWISS MODEL (http://swissmodel.
expasy.org/SWISS-MODEL.html) [36] which predicts and
constructs the three-dimensional structure according to the
sequence homologies. Using insight ii software (Accerlys,
San Diego, CA), the model of SIK2 was superimposed on
Chk1 from Chk1–staurosporine complex, the structure of
which had already been determined (PDBID 1NVR) [37],
and the staurosporine was placed at the corresponding
position of the SIK1 to the staurosporine-binding site on
the Chk1. After energy minimization of the complex model,
we obtained a final model of the SIK2–staurosporine com-
plex. We next calculated the electrostatic potential on the
molecular surface [38] of SIK2 using the program SCB
developed by Nakamura & Nishida [39]. The illustration
Y. Katoh et al. Silencing of CREB by LKB1-SIK
FEBS Journal 273 (2006) 2730–2748 ª 2006 The Authors Journal compilation ª 2006 FEBS 2745
was prepared using molfeat v2.2 (FiatLux Co. Ltd,
Tokyo, Japan).
Acknowledgements

We are grateful to Dr Marc R. Montminy (Salk Insti-
tute, USA), Dr K. Morohashi (National Institute for
Basic Biology, Japan) and Dr T. Sugawara (Hokkaido
University, Japan) for providing us the EXV-1,
CYP11A and StAR reporter plasmids, respectively.
These plasmids were used in the original version of this
manuscript. We thank Dr D. Carling (MRC Clinical
Sciences Centre, UK) for critical evaluation of our
data. The cDNAs for SIK3 and MARK4 were gifts
from the Kazusa DNA Institute, Japan. This study
was supported by Grants-in-Aid for Scientific Research
from the Ministry of Education, Culture, Sports, Sci-
ence and Technology and Ministry of Health, Labor
and Welfare Japan, and a grant from the Technology
Research Grant Program in ‘03’ from the New Energy
and Industrial Technology Development Organization
(NEDO) of Japan.
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Supplementary material
The following material is available online:
Fig. S1. (A) Dose-dependent inhibition of staurospo-
rine against the phorbol ester-induced AP-1 activity
(PKC activity) in HEK293 cells. HEK293 cells that
had been transformed with 0.2 lg of reporter plasmids,
pTA-AP1 (PMA) and 0.03 lg of phRL-TK(Int
-
), were
treated with phorbol myristate acetate (10 nm) for 6 h
in the presence of the indicated concentrations of sta-
urosporine. (B) Dose-dependent inhibition of the pep-
tide phosphorylation activity of SIK1 or AMPK by
staurosporine in vitro. The GST–TORC2 peptide
(4 lgÆmL
)1
) was coated on 96-well plates. After block-

ing the plates with BlockAce reagent (Yukizirushi Co.
Ltd, Tokyo Japan), 0.1 ng of GST–SIK1 (1–354) and
0.05 ng of AMPKa (purified from rat liver: Upstate,
Dundee, UK) was added into the wells with reaction
buffer (50 mm Tris ⁄ HCl, pH ¼ 7.4, 10 mm MgCl
2
,
Y. Katoh et al. Silencing of CREB by LKB1-SIK
FEBS Journal 273 (2006) 2730–2748 ª 2006 The Authors Journal compilation ª 2006 FEBS 2747
0.1 mm ATP and 0–10 nm staurosporine) and incuba-
ted for 1 h. The phospho-Ser171 was detected by the
anti-(phospho-Ser171 IgG) ⁄ peroxidase-conjugated anti-
(rabbit IgG) using ABTS-substrate. We measured opti-
cal density at 405 nm. The densities of the wells with-
out enzyme (background) and without staurosporine
were converted to the points at 100 and 0% inhibition,
respectively. (C) Dose-dependent upregulation of the
basal CRE-reporter activity (black) and the inhibition
of the SIK1-dependent inhibition of the forskolin-
induced CRE activity (blue). The ‘SIK1-dependent
inhibition of forskolin-induced CRE activity’ is
expressed as the difference between the CRE activity
in the presence of forskolin alone minus that with
SIK1 (S577A) n ¼ 3. (D) The specificity of the anti-
(phospho-Thr182 (SIK1) IgG) was evaluated by an in
vitro kinase assay. The GST-fusion A-loop peptide,
WT or T182A, was prepared using E. coli (right) and
phosphorylated by the LKB1 complex (left). The phos-
phorylated peptide was specifically recognized by the
anti-(pT182 IgG).

This material is available as part of the online article
from
Silencing of CREB by LKB1-SIK Y. Katoh et al.
2748 FEBS Journal 273 (2006) 2730–2748 ª 2006 The Authors Journal compilation ª 2006 FEBS